The FMS-like tyrosine kinase (FLT3) is a type III receptor tyrosine kinase that is thought to play a key role in hematopoiesis. Certain classes of FLT3 mutations cause constitutively activated forms of the receptor that are found in significant numbers of patients with acute myeloid leukemia (AML). The mutations occur in either the activation loop, for example, as point mutations of Asp835, or as internal tandem duplication (ITD) sequences in the juxtamembrane (JM) domain.
FLT3 (FMS-like tyrosine kinase 3) (Gilliland, D. G., and Griffin, J. D., Blood 100: 1532-1542 (2002); D. Kottaridis, P., et. al., Br. J. Haematol. 122, 523-538 (2003); Stirewalt, D. L. and Radich, J. P. Nat. Rev. Cancer 3: 650-665 (2003)), also known as FLK-2 (fetal liver kinase 2) and STK-1 (human stem cell kinase 1), belongs to a family of type III receptor tyrosine kinases (RTKs) (Rosnet, O., et. al., Oncogene 6: 1641-1650 (1991); Rosnet, O., et. al., Genomics 9: 380-385 (1991); Small, D., et. al., Proc. Natl. Acad. Sci. USA 91, 459-463 (1994); Matthews, W., et. al., Cell 65: 1143-1152 (1991)). Members of a subset of this family include FLT3, platelet-derived growth factor receptors α and β (PDGFRα and PDGFRβ) (Yarden, Y., et. al., Nature 323: 226-232 (1986); Claesson-Welsh, L., et. al., Methods Enzymol. 198: 72-77 (1991); Claesson-Welsh, L et. al., Proc. Natl. Acad. Sci. USA 86: 4917-4921 (1989); Matsui, T., et. al., Science 243: 800-804 (1989)), FMS (Stanley, E. R., et. al., J. Cell. Biochem. 21:151-159 (1983)) and cKIT (Yarden, Y et. al., EMBO J. 6: 3341-3351 (1987); Mol, C. D., et. al., J. Biol. Chem. 278: 31461-31464 (2003)) and are characterized by an extracellular domain consisting of five immunoglobulin-like (Ig-like) domains, a single transmembrane region, a cytoplasmic juxtamembrane domain (JM) and a cytoplasmic tyrosine kinase domain interrupted by a kinase insert domain (KID) (Agnes, F., et. al., Gene 145: 283-288 (1994); Rosnet, O., and Birnbaum, D. (1993) Crit. Rev. Oncog. 4, 595-61; Scheijen, B., and Griffin, J. D. Oncogene 21: 3314-3333 (2002)). Two groups independently reported the cloning of the flt3 gene (Rosnet, O., et. al., Oncogene, supra; Rosnet, O., et. al., Genomics, supra; Matthews, W et. al., supra).
Subsequently, FL, the ligand for FLT3, and a type I transmembrane protein was cloned from mouse (Lyman, S. D., et. al., Stem Cells 12 Suppl 1: 99-107; discussion 108-110 (1994); Lyman, S. D et. al., Oncogene 11: 1165-1172 (1995); Hannum, C., et. al., Nature 368: 643-648 (1994); Savvides, S. N., et. al., Nat. Struct. Biol. 7: 486-491 (2000)). The binding of FL leads to dimerization, activation and autophosphorylation of the receptor and subsequent activation of several signaling pathways including STAT5 (Zhang, S., et. al., J. Exp. Med. 192: 719-728 (2000)), Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)/AKT pathways. The human flt3 gene encodes a 993 amino acid protein of which residues 572-603 and 604-958 represent the JM and tyrosine kinase domains, respectively (Rosnet, O., et. al., Blood 82: 1110-1119 (1993)).
FLT3 is primarily expressed in immature hematopoietic cells (Rosnet, O., et. al., Genomics, supra; deLapeyriere, O., et. al., Differentiation 58: 351-359 (1995)) and is essential for the normal function of stem cells and the immune system (deLapeyriere, O., et. al., supra; Brasel, K., et. al., Leukemia 9: 1212-1218 (1995); Turner, A. M., et. al., Blood 88: 3383-3390 (1996)). FLT3 is also found in placenta, gonads and brain (Maroc, N., et. al., Oncogene 8: 909-918 (1993)) and is expressed in high levels in a wide range of hematopoietic malignancies including 70-100% of acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia (Rosnet, O., et. al., Acta Haematol. 95: 218-223 (1996); Drexler, H. G. Leukemia 10: 588-599 (1996)).
Two distinct types of FLT3 mutations have been identified in up to 41% of AML patients. Internal tandem duplication (ITD) mutations within the JM domain contribute to about 17-34% of FLT3 activating mutations in AML (Nakao, M., et. al., Leukemia 10: 1911-1918 (1996); Thiede, C., et. al., Blood 99: 4326-4335 (2002)). FLT3-ITD has also been detected at low frequency in myelodysplastic syndrome (MDS) (Yokota, S., et. al., Leukemia 11: 1605-1609 (1997)); Horiike, S., et. al., Leukemia 11: 1442-1446 (1997)). The ITDs are always in-frame, and are limited to the JM domain. However, they vary in length and position from patient to patient. These repeat sequences may serve to disrupt the autoinhibitory activity of the JM domain resulting in the constitutive activation of FLT3. Point mutations at aspartate 835 within the activation loop of the FLT3 kinase domain represent a second class of activating mutations (Yamamoto, Y., et. al., Blood 97: 2434-2439 (2001); Abu-Duhier, F. M., et. al., Br. J. Haematol. 113: 983-988 (2001)). FLT3-Asp835 mutations also lead to constitutive activation of the receptor and have been reported in 7% of AML, 3% of MDS and 3% of all cases. The most common substitution is Asp835Tyr, but other substitutions including Asp835Val, Asp835His, Asp835Glu and Asp835Asn have also been reported (Yamamoto, Y., et. al., supra). Both FLT3-ITD and FLT3-Asp835 mutations are associated with FLT3 autophosphorylation and phosphorylation of downstream targets (Yamamoto, Y., et. al., supra; Mizuki, M., et. al., Blood 96: 3907-3914 (2000); Mizuki, M., et. al., Blood 101: 3164-3173 (2003); Hayakawa, F., et. al., Oncogene 19: 624-631 (2000)).
A novel class of constitutively activated FLT3 mutants has been recently identified in AML patients in which isoleucine 836 is either deleted (FLT3-Ile836del) or substituted with methionine and arginine (FLT3-Ile836Met+Arg) (Thiede, C., et. al., supra). In mice, injection of FLT3-ITD transformed cells results in leukemia-like syndrome (Mizuki, M., et. al., (2000), supra). Several FLT3 inhibitors, such as PKC412 (N-benzoyl staurosporine) (Fabbro, D., et. al., Anticancer Drug Des. 15: 17-28 (2000); Weisberg, E., et. al., Cancer Cell 1: 433-443 (2002)), CT53518 (also known as MLN518) (Kelly, L. M., et. al., Cancer Cell 1: 421-432 (2002)), SU11248 (O'Farrell, A. M., et. al., Blood 101: 3597-3605 (2003)), SU5614 (Spiekermann, K., et. al., Blood 101: 1494-1504 (2003)), and SU5416 (Giles, F. J., et. al., Blood 102: 795-801 (2003)), have been shown to have antitumor activity. Collectively, these data suggest that FLT3 is an attractive therapeutic target for the development of kinase inhibitors for AML and other associated diseases.